Concern has been raised that altering the fraction of inspired O₂ (Fi(O₂)) could accelerate or decelerate microbubble dissolution time within the pulmonary vasculature and thereby invalidate the ability of saline contrast echocardiography to detect intrapulmonary arteriovenous shunt in subjects breathing either a low or a high Fi(O₂). The present study determined whether the gaseous component used for saline contrast echocardiography affects the detection of exercise-induced intrapulmonary arteriovenous shunt under varying Fi(O₂). Twelve healthy human subjects (6 men, 6 women) performed three 11-min bouts of cycle ergometer exercise at 60% peak O₂ consumption (Vo(2peak)) in normoxia, hypoxia (Fi(O₂) = 0.14), and hyperoxia (Fi(O₂) = 1.0). Five different gases were used to create saline contrast microbubbles by two separate methods and were injected intravenously in the following order at 2-min intervals: room air, 100% N₂, 100% O₂, 100% CO₂, and 100% He. Breathing hyperoxia prevented exercise-induced intrapulmonary arteriovenous shunt, whereas breathing hypoxia and normoxia resulted in a significant level of exercise-induced intrapulmonary arteriovenous shunt. During exercise, for any Fi(O₂) there was no significant difference in bubble score when the different microbubble gas compositions made with either method were used. The present results support our previous work using saline contrast echocardiography and validate the use of room air as an acceptable gaseous component for use with saline contrast echocardiography to detect intrapulmonary arteriovenous shunt during exercise or at rest with subjects breathing any Fi(O₂). These results suggest that in vivo gas bubbles are less susceptible to changes in the ambient external environment than previously suspected.